<i>Background:</i> Asthma and chronic obstructive pulmonary disease (COPD) display features of overlap in airway physiology and airway inflammation. Whether inflammatory phenotypes in airway disease describe similar mediator expression is unknown. <i>Objectives:</i> To explore the relationship of airway inflammation and cytokine and chemokine expression in asthma and COPD. <i>Methods:</i> Subjects with asthma and COPD (n = 54 and n = 49) were studied. Clinical characteristics and sputum were collected at entry into the study. A 2-step sputum processing method was performed for supernatant and cytospin preparation. Meso Scale Discovery and Luminex platforms were used to measure cytokines, chemokines and matrix metalloproteinase levels. <i>Results:</i> Analytes sensitive to dithiothreitol (DTT) that had increased recovery in the 2-step sputum process were IL-1β, 4, 5, 10, 13, IFN-γ, TNFRI, GM-CSF, CCL2, 3, 4, 5, 13 and 17. There was a differential expression in IL-8, TNFRI and TNFRII between asthma and COPD [mean fold difference (95% CI): IL-8, 2.6 (1.3–5.4), p = 0.01; TNFRI, 2.1 (1.3–5.4), p = 0.03; TNFRII, 2.6 (1.2–5.6), p = 0.02]. In neutrophilic and eosinophilic airway inflammation, TNFα, TNFRI, TNFRII, IL-6, IL-8 and IL-5 could differentiate between these phenotypes. However, these phenotypes were unrelated to the diagnosis of asthma or COPD. <i>Conclusion:</i> Recovery of sputum mediators sensitive to DTT can be improved using the described sputum processing technique. Within airway inflammatory sub-phenotypes there is a differential pattern of mediator expression that is independent of disease. Whether these inflammatory phenotypes in asthma and COPD confer distinct pathogeneses, therapeutic responses and clinical phenotypes needs to be further evaluated.